Project Summary Age-related macular degeneration (AMD) is the primary cause of blindness in the developed world, and as populations age, AMD will become an increasingly large burden on our health care system. The eye represents one of the few tissues amenable to current gene therapy techniques, and thus identifying specific genetic changes giving rise to AMD would be a critical step in developing AMD-correcting gene therapies. Toward this end, recent genetic association studies have identified a genomic region that represents a major risk locus for AMD. The risk alleles associated with AMD in this region are characterized by significant and large odds ratios (>5), implicating this as a major determinant of disease susceptibility. The AMD-associated region lies at chromosomal location 10q26. The region encompasses all or part of three genes, HTRA1, ARMS2, and PLEKHA1, all of which are candidates to affect the retina and be a causal factor in AMD development. Further, the nature of genotyping data used in the association studies means that the associated SNPs are actually ?sentinel? SNPs representing a large haplotype block containing many other variants. It is therefore difficult to assess which SNP or indel is actually responsible for any functional effect contributing to the disease. A recent resequencing effort has identified at least 67 variants located within the AMD-associated locus at 10q26, any or many of which could be functional. Limited functional studies in the region have led to conflicting reports regarding which variant sites are functional, and which gene is the target of those sites, with some evidence supporting either or both of HTRA1 and ARMS2 as the key functional target. To efficiently assay and identify functional variants in such large genomic regions, the lab has developed new technologies for the assembly of very large DNA molecules. These techniques have been used to date for assembling megabase-sized yeast chromosomes, as well as myriad metabolic pathways and human gene loci. Further, methods for efficient incorporation of these large assemblies into the genome of various mammalian cell types have been described. The assembly strategy for these so-called ?assemblons? allows combinatorial incorporation of variable segments, thus enabling systematic functional analysis of single or multiple variants in parallel. The proposed study will utilize these technologies together to rapidly identify and characterize the in vitro and in vivo effects of AMD-associated variants in cells. Identification of the causal variants will enable targeted genotyping for AMD diagnosis and eventually development of targeted gene therapy to ameliorate AMD symptoms.